A major objective of the proposed research is to correlate the structure and function of human thyroxine-binding globulin (TBG), physiologically the most important carrier of thyroid hormone in human blood. The method of equilibrium dialysis will be used to obtain further data on the interaction of TBG with thyroxine (T4) and triiodothyronine (T3). The T4-binding site on TBG will be investigated chemically in order to identify amino acid residues which take part in binding. Based on the principal of affinity labeling, tritiated compounds capable of reaction with side chains and structurally related to 1-anilino-8-naphthalenesulfonate (a known competitor for the T4-binding site) will be used as chemical probes of the binding site. TBG will also be derivatized by reaction with various group selective reagents which are known to react with histidyl, tyrosyl and lysyl groups and the resultant effect on binding will be determined. The transport of thyroid hormones will be studied in a model homologous system containing purified human TBG and human adipose, a known target tissue. Uptake of I125-T4 (or I125-T3) into adipose cells in the presence and absence of TBG will be studied to note the effect of TBG on transport. Doubly labeled, I125-T4-I131-TBG will be used to determine if the T4-TBG complex can be taken up directly by cells. It should be possible to obtain a definitive answer to the question whether T4 (or T3) enters cells only by a process of passive diffusion or whether specific binding sites for free T4 or TBG (or the TBG-T4 complex) exist on the cell surface. The uptake of injected I125-T3 into cytosol and nuclear fractions of bullfrog tadpoles at different stages of metamorphosis will be studied to ascertain whether the induction of specific binding proteins or receptors for T3 may occur in liver.